Regulated gene expression has utility in a variety of applications including the expression of recombinant proteins, modified production of various metabolites, functional studies in cell-based assays and in vivo in transgenic animals, in gene therapy vectors, and in plant expression vectors for controlled transgene expression.
Gene therapy is a fast evolving area of medical and clinical research. Gene therapy encompasses gene correction therapy and transfer of therapeutic genes and is being applied for treatment of cancer, infectious diseases, monogenic diseases, multigenic diseases, and acquired diseases.
There are an increasing number of anecdotal cases of efficacy in the use of gene therapy for the treatment of monogenic diseases, early stage tumors, and cardiovascular disease (Blaese, et al., 1995; Wingo, et al., 1998; Dzau, et al., 1998; Isner, et al., 1998). However, all of the currently utilized methods of gene transfer typically demonstrate low transfer efficiency and expression rates. As the technology is improved and high efficiency gene transfer and expression is achieved, the ability to regulate such expression on both a temporal and spatial level becomes increasingly important.
In addition, the development of plants having desired traits such as improved yield; disease resistance to fungal, bacterial, viral and other pathogens; insect resistance; improved fruit ripening characteristics; cold temperature and dehydration tolerance; increased salt and drought tolerance; improved food quality (i.e., nutritional content) and improved appearance has been the focus of agribusiness for many years. At present, the regulated expression of transgenes in plants with optimal expression of target genes in manner that does not result in harm to the plant is the focus of extensive research.
Attempts to control gene activity have been made using various inducible eukaryotic promoters, such as those responsive to heavy metal ions, heat shock or hormones. In most cases, the effect of exogenous inducers is pleiotropic, in that it induces the expression of endogenous cellular genes in addition to the target transgene. Second, many promoter systems exhibit high levels of basal activity in the non-induced state, i.e., endogenous activators often interfere with regulation of transgene expression.
Several systems for regulatable expression of genes (“gene switch” systems) have been reported in the literature. Such systems are based on modifying the activity of synthetic regulatory proteins, which bind to double stranded DNA and control the activity of a promoter for a given gene, by the use of exogenous inducers (compounds) that specifically interact with a particular synthetic regulatory protein.
In systems where an inducer interacts with a regulatory protein, the regulatory protein dictates the selection of inducer. So, the ability to choose an inducer with better pharmacological properties are limited by the selection of regulatory protein.
Methods for screening and constructing molecules, which have properties of sequence specific DNA binding and displacement of protein that is bound at flanking or adjacent sites on a DNA sequence, have been reported in co-owned U.S. Pat. Nos. 5,306,619, 5,693,463, 5,716,780, 5,726,014, 5,744,131, 5,738,990, 5,578,444, 5,869,241.
Using such methods, several classes of small molecules that interact with double-stranded DNA have been identified, and shown to preferentially recognize specific nucleotide sequences.
A need exists for the development of systems for regulatable gene expression which are controlled, inducible by compounds targeted to polynucleotides, and characterized by low toxicity and favorable pharmacokinetic properties.